Cervix cancer is one of the most frequent malignant tumors in women. Incidence of invasive cervix cancer is slowly decreasing, but it is still one of the most frequent cancers that takes 25% of total woman cancer in the developmental countries (Harro et al, J Natl Cancer Inst 93(4):284-292, 2001).
Clinical and molecular epidemiological studies say human papilloma virus (referred as “HPV” hereinafter) infection is the major cause of cervix cancer (Brisson et al, Am J Epidemiol 140(8):700-710, 1994; Schiffman et al, J Natl Cancer Inst 85(12):958-964, 1993). HPV is a small DNA virus composed of approximately 8000 nucleotides and causing benign and malignant tumors. Up to date, 100 or more HPV subtypes have been classified according to genome and genotypes of approximately 90 HPV subtypes have been completely analyzed. Among these, high risk HPV types (for example, HPV-16, 18, 31, 33, 35, 45, 51, 52 and 56) are involved in almost 90% of cervix cancer cases. Among cervix cancers caused by HPV infection, at least 50% are associated with HPV-16, and HPV-18 (12%), HPV-45 (8%), and HPV-31 (5%) follow (Munoz and Bosch, Salud Publica Mex 39(4):274-282, 1997).
HPV encodes two oncoproteins, protein E6 and protein E7, which are involved in cell immortalization and transformation via HPV. Oncoprotein E6 is bound to tumor suppressor protein p53 to decompose the p53 through ubiquitin pathway. In the meantime, oncoprotein E7 is directly bound to Rb to induce hyper-phosphorylation (Dyson et al, Science 243(4893):934-937, 1989; Huibregtse et al, Mol Cell Biol 13(2):775-784, 1993a; Huibregtse et al, Mol Cell Biol 13(8):4918-4927, 1993b; Munger et al, Embo J 8(13):4099-4105, 1989). First, protein E6 forms a complex with E6-AP (E6-associated protein) that is E3 ubiquitin-protein ligase. Then, the E6/E6-AP complex is combined with wild type p53 to induce ubiquitination, suggesting that the complex interrupts p53 mediated cell response to DNA damage.
The tumor suppressor protein p53 is regulated by Mdm2-mediated ubiquitination. However, in the case of cervix cancer with HPV infection, p53 decomposition is accomplished by ubiquitination mediated not by Mdm2 but by E6 (Hengstermann et al, Proc Natl Acad Sci U S A 98(3):1218-1223, 2001).
Therefore, unlike many other cancers, cervix cancer with HPV infection exhibits has wild type p53 gene (Hainaut et al, Nucleic Acids Res 26(1):205-213, 1998; Scheffner et al, Proc Natl Acad Sci U S A 88(13):5523-5527, 1991). However, the expression level of the protein p53 therein is very low because it is decomposed by protein E6.
In particular, HPV E6 is a promising target for the treatment of cervix cancer. Approaches targeting E6 or E6/E6-AP complex result in various treatment methods.
For example, attempts using cytotoxic agents, anti-viral agents releasing Zn of the oncoprotein E6, epitope-peptides (mimotope) of E6-AP, anti-E6 lybozymes, peptide aptarmers targeting the viral oncoprotein E6, siRNAs targeting the viral oncoprotein E6 gene, and co-use thereof, etc (Beerheide et al, J Natl Cancer Inst 91(14):1211-1220, 1999; Beerheide et al, Bioorg Med Chem 8(11):2549-2560, 2000; Butz et al, Proc Natl Acad Sci USA 97(12):6693-6697, 2000; Butz et al, Oncogene 22(38):5938-5945, 2003; Jiang and Milner, Oncogene 21(39):6041-6048, 2002; Liu et al, Biochemistry 43(23):7421-7431, 2004; Wesierska-Gadek et al, Int J Cancer 101(2):128-136, 2002; Yoshinouchi et al, Mol Ther 8(5):762-768, 2003; Zheng et al, Di Yi Jun Yi Da Xue Xue Bao 22(6):496-498, 2002) have been made.
It has been recently proved that siRNA not only can silence a specific endogenous gene selectively in animal cells (Sui et al, Proc Natl Acad Sci U S A 99(8):5515-5520, 2002; Yu et al, Proc Natl Acad Sci U S A 99(9):6047-6052, 2002) but also can silence a viral gene in the case of the virus mediated disease (Ge et al, Proc Natl Acad Sci U S A 100(5):2718-2723, 2003; Kitabwalla and Ruprecht, N Engl J Med 347(17):1364-1367, 2002; Milner, Expert Opin Biol Ther 3(3):459-467, 2003).
RNA interfering induced by siRNA transfection draws our attention as a new therapeutic method for virus infection in human.
siRNA that targets E6 and E7 in cervix cancer cells infected with HPV accumulates p53 and pRb, leading to apoptosis or senescence. RNAi targeting E6 and E7 has been confirmed to silence the expressions of these proteins in the cervix cancer cell line infected with HPV-16 (Jiang and Milner, Oncogene 21(39):6041-6048, 2002; Putral et al, Mol Pharmacol 68(5):1311-1319, 2005; Yoshinouchi et al, Mol Ther 8(5):762-768, 2003) and in the cell line infected with HPV-18 (Butz et al, Oncogene 22(38):5938-5945, 2003; Gu et al, Cancer Gene Ther, 2006; Hall and Alexander, J Virol 77(10):6066-6069, 2003). In spite of the above results, the methods are still in the middle of controversy because they cause low growth, senescence or apoptosis.
A paper describing combination therapy of chemotherapy using cisplatin and radiotherapy was published in 1999 (Thomas G M, N Engl J Med. 340(15):1198-1200, 1999). This method could significantly improve survival rate of women with severe local cervix cancer. Cisplatin is a DNA damaging drug which is widely used for the treatment of ovarian cancer, cervix cancer, head cancer, neck cancer, non-small cell lung cancer, etc. Most recently, the mechanism of this drug was precisely investigated based on platinum. However, the mechanism including the processes of absorption and excretion of the drug, signal transduction of DNA damage, cell cycle arrest, DNA repair and apoptosis has not been disclosed, yet (Wang and Lippard, Nat Rev Drug Discov 4(4):307-320, 2005).
In HPV-18 HeLa cells, after the treatment with cisplatin, p53 is released from the E6 mediated degradation pathway and preferentially accumulated in nucleus (Wesierska-Gadek et al, Int J Cancer 101(2):128-136, 2002). In HPV-16 SiHa cells, combination therapy of radiotherapy and cisplatin results in the recovery of p53 functions, so that sensitivity to radiation is increased (Huang et al, J Cell Biochem 91(4):756-765, 2004).
In cells infected with high risk HPV, combination therapy of siRNA targeting E6 and chemotherapy with cisplatin or radiotherapy is expected to bring cytotoxic effect, based on the theory that E6 siRNA acts as an effective chemical or radiation sensitizer.
However, other researches showed such results that the transcript that does not have 100% complementarity with siRNA can also induce gene silencing by RNA interference (Fedorov et al, Rna 12(7):1188-1196, 2006), which is called “off-target effect”. That is, RNA interference is not authentically specific to a target, so that non-target genes can be silenced according to the concentration of siRNA. Nevertheless, no previous studies have reported such off-target effect when they carried out experiments with siRNA against HPV.
Therefore, the present inventors completed this invention by confirming that combination therapy of siRNA having the sequence specific to HPV E6 and low concentration of cisplatin could result in anticancer effect in cervix cancer cells.